Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry.

BACKGROUND: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. AIM: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. METHODS: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn ADP Flow Cytometer. RESULTS: The pHi clamping conditions were optimized as 140 mM potassium and 10 microM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. CONCLUSION: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.     

Identification of recurrent regions of copy-number variants across multiple individuals

Background  

Algorithms and software for CNV detection have been developed, but they detect the CNV regions sample-by-sample with individual-specific breakpoints, while common CNV regions are likely to occur at the same genomic locations across different individuals in a homogenous population. Current algorithms to detect common CNV regions do not account for the varying reliability of the individual CNVs, typically reported as confidence scores by SNP-based CNV detection algorithms. General methodologies for identifying these recurrent regions, especially those directed at SNP arrays, are still needed.     

A New Benzoylphloroglucinol Derivative with an Adamantyl Skeleton and Other Constituents from Garcinia multiflora: Effects on Neutrophil Pro‐Inflammatory Responses

A novel benzoylphloroglucinol derivative, garcimultiflorone D ( 1 ), with an unusual adamantyl‐caged skeleton was isolated from the fruits of Garcinia multiflora, together with four known compounds. The structure of 1 was determined through extensive 1D/2D‐NMR and mass‐spectrometric analyses. Garcimultiflorone D ( 1 ) exhibited inhibitory activities with IC₅₀ values of 7.21±1.07 and 6.01±0.37 μg/ml against fMLP/CB‐induced superoxide anion generation and elastase release, respectively.     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.     

Spermatogenesis of a marine snail,Littorina sitkana

Summary The fine structure of the spermatogonium, spermatocyte and spermatid of a marine snail, Littorina sitkana is described. The ring centriole (annulus) is formed from the distal centriole and it migrates to the base of the mitochondrial region where it lies in a joint-like structure which is formed by an area of invaginated plasma membrane. The distal and proximal centrioles are at first perpendicular to each other but the proximal centriole rotates to a position coaxial with the distal centriole and fuses with it. The peripheral doublet fibers are continuous between the two centrioles but the central fibers originate only in the distal centriole. The acrosome differentiates from the proacrosomal granule which is derived from a Golgi body. Microtubules, present at this stage, may assist acrosomal formation. Chromatin condensation begins with the formation of fibrous strands, then to lamellar plates which become folded and later twisted around the flagellar shaft. In the final stages the lamellae appear in cross section as concentric rings which eventually fuse to form a homogeneously dense nuclear tube.     

Base composition studies on mitochondrial 4 S RNA from rat liver and Morris hepatomas 5123D and 7777.

The major and modified base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method. The base composition of these mitochondrial 4 S RNA preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs. The results of these studies are: 1. When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are characterized by high (A + U)/(G + C) ratios and low overall degrees of base methylation and modification. 2. The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4-S RNAs than from their cytoplasmic counterparts. Thus, several modified constituents found in both cytoplasmic and mitochondrial 4 S RNA are absent from the bacterial 4-S RNAs. 3. Mitochondrial 4S RNA from both hepatomas was found to be under-methylated and undermodified when compared with normal liver mitochondrial 4S RNA. This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepatoma 5123D (i.e., 8% undermethylation). These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors.     

秦岭川金丝猴一周岁内个体的行为发育

2003年3月至2004年5月在陕西周至国家自然保护区的玉皇庙地区,采用目标动物取样法(Focal ani-mal sampling)对2003年出生的5只金丝猴个体行为发育进行了观察,应用全事件记录法(All-occurrences record-ing)进行数据的收集。结果表明23日龄婴儿首次从母亲怀内爬出在树枝上活动,68日龄开始在树上攀爬跳跃,5月龄主动采食树叶和啃投食区食物,6月龄跟随社群迁移。依据婴幼儿主要行为首次出现时间及其变化,把1周岁内个体发育分为5个时期,即完全依赖期、探索外部世界期、融入社群期、适应生存期和逐步独立期。1周岁内婴幼儿活动和休息的地方是母亲怀里、其它个体怀里、树枝上和地面上。随着发育,婴幼儿在这些地方停留的时间也在变化。1周岁内婴幼儿在母亲怀中的时间与年龄存在明显的负相关,在其它个体怀里的时间与年龄也呈显著负相关。虽然独自在树上的时间与年龄相关性不显著,但在地面上停留的时间与年龄呈正相关。     

Maculatin 1.1, an anti-microbial peptide from the Australian tree frog, Litoria genimaculata solution structure and biological activity.

The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.     

Drosophila neural progenitor polarity and asymmetric division

In the Drosophila embryonic central nervous system, the neural precursor cells called neuroblasts undergo a number of asymmetric divisions along the apical-basal axis to give rise to different daughter cells of distinct fates. This review summarizes recent progress in understanding the mechanisms of these asymmetric cell divisions. We discuss proteins that are localized at distinct domains of cortex in the neuroblasts and their role in generating asymmetry. We also review uniformly cortical localized factors and actin cytoskeleton-associated motor proteins with regard to their potential role to serve as a link between distinct cortical domains in the neuroblasts. In this review, asymmetric divisions of sensory organ precursor and larval neuroblasts are also briefly discussed.     

Enhanced enterovirus 71 virus‐like particle yield from a new baculovirus design

Enterovirus 71 (EV71) is responsible for the outbreaks of hand‐foot‐and‐mouth disease in the Asia‐Pacific region. To produce the virus‐like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co‐express EV71 P1 polypeptide and 3CD protease using the Bac‐to‐Bac^® vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD? vector system which was deficient in v‐cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD? system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF‐P1‐C3CD, a recombinant baculovirus constructed using the flashBAC GOLD^TM system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High Five^TM cells with BacF‐P1‐C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 μg purified VLP induced cross‐protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 μg VLP, one liter High Five^TM culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines. Biotechnol. Bioeng. 2015;112: 2005–2015. © 2015 Wiley Periodicals, Inc.